Crystal modification of a N-phenyl-2-pyrimidineamine derivative, processed for its manufacture and its use

ABSTRACT

The invention relates to a new crystalline form of the methanesulfonic acid addition salt of 4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]-benzamide of formula 1, which may be used for example for tumour therapy.

The invention relates to a particular form of the methanesulfonic acidaddition salt of4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyrimidin-3-yl)pyrimidin-2-ylamino)phenyl]-benzamide,comprising certain crystals, processes for the preparation thereof,pharmaceutical compositions containing this crystal form, and their usein diagnostic methods or preferably for the therapeutic treatment ofwarm-blooded animals, especially humans, or their use for thepreparation of pharmaceutical preparations for use in diagnostic methodsor preferably for the therapeutic treatment of warm-blooded animals,especially humans.

BACKGROUND TO THE INVENTION

The preparation of4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]benzamideand the use thereof, especially as an anti-tumour agent, are describedin Example 21 of EP-A-0 564 409, which was published on 6 Oct. 1993, andin equivalent applications in numerous other countries. This compound isexemplified in these publications only in free form (not as a salt).

It has now been surprisingly found that a crystal form may under certainconditions be found in the methanesulfonate salt of this compound, whichis described hereinafter as β-crystal form, and which has veryadvantageous properties.

DETAILED DESCRIPTION OF THE INVENTION

The invention is described in more detail in the following with the helpof drawings and other aids:

DESCRIPTION OF THE DRAWINGS

FIG. 1/3 shows the X-ray diffraction diagram of the α-crystal form ofthe methanesulfonic acid addition salt of a compound of formula I.

FIG. 2/3 shows the X-ray diffraction diagram of the β-crystal form ofthe methanesulfonic acid addition salt of a compound of formula I.

FIG. 3/3 shows the crystals above of the α-crystal form and below of theβ-crystal form of4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]-benzamidemethanesulfonate (=of the methanesulfonic acid addition salt of acompound of formula I).

In both X-ray diagrams, the angle of refraction 2theta is plotted on thehorizontal axis (x-axis) and the relative line intensity(background-corrected peak intensity) on the vertical (y-axis). Thediagrams are obtained as follows: first, the x-ray diffraction diagramis recorded on film using a Guinier camera (Enraf-Nonius FR 552 model)with a Guinier 258-94c film and copper radiation (Kα1 radiation,wavelength λ=1.54060 Angström). The optical density of the lines on thefilm is proportional to the light intensity. The film is then scanned inusing a line scanner (LS 18, Johansson, Täby, Sweden) with SCANPIsoftware.

In accordance with FIG. 2/3 there are lines having a relative lineintensity of 20 or more at the following angles of refraction 2theta(relative line intensities given in parentheses): 9.7° (40), 13.9° (26),14.7° (23), 17.5° (57), 18.2° (90), 20.0° (65), 20.6° (76), 21.1° (100),22.1° (89), 22.7° (38), 23.8° (44), 29.8° (23) and 30.8° (20). The factthat in FIG. 2/3 the relative line intensity of the line at 30.8° seemsto be higher than that of the line at 29.8° is due to a close by furtherline at 31.0° having a relative line intensity of 13.

Melting points are determined by means of a DSC thermogram using aMettler-Toledo TA8000. DSC (“differential scanning calorimetry”) is thetechnique of dynamic differential calorimetry. Using this technique, themelting temperature both of the α-crystal form and of the β-crystal formcan be measured by heating the samples until a thermal, i.e. anendothermic or exothermic, reaction is detected by means ofultrasensitive sensors. The melting points indicated in this text aredetermined using a Mettler-Toledo TA8000 apparatus, about 5.5 to 6.5 mgof each sample being measured in an aluminium crucible with a perforatedlid under a quiescent atmosphere of air at a heating rate of 10° C./min(starting at 20° C.).

The α-crystal form of4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]benzamidemethanesulfonate is characterised by needle-shaped crystals and ishygroscopic. In this form, the crystals are not particularly well-suitedto pharmaceutical formulation as solid dosage forms, because theirphysical properties, for example their flow characteristics, areunfavourable. Under certain conditions, however, it is possible toobtain4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]benzamidemethanesulfonate in a crystal form which is not needle-shaped. This formis described in the present text as β-crystal form.

The β-crystal form of4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]benzamidemethanesulfonate has the advantage that its flow properties aresubstantially more favourable than those of the α-crystal form. Thiscrystal form has the further advantage of being thermodynamically morestable at temperatures below 140° C. Finally, the β-crystal form is lesshygroscopic than the α-crystal form and thus also stores better and iseasier to process.

The invention relates to an acid addition salt of a compound of formulaI comprising non-needle-shaped crystals, especially the β-crystal formof the methanesulfonic acid addition salt of the compound of formula I.

The invention relates especially to a particular, essentially purecrystal form, preferably that which is referred to hereinafter as theβ-crystal form, of the methanesulfonic acid addition salt of4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyrid-3-yl)pyrimidin-2-ylamino)phenyl]benzamidemethanesulfonate of formula I,

Where the term methanesulfonic acid salt of a compound of formula I orof4-(4-methyl-piperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]benzamideis used hereinbefore and hereinafter, this is especially taken to meanthe methanesulfonic acid salt of formula II.

The term“essentially pure” is understood in the context of the presentinvention to mean especially that at least 90, preferably at least 95,and most preferably at least 99 per cent by weight of the crystals of anacid addition salt of formula I are present in the crystal formaccording to the invention, especially the β-crystal form,

In the context with stating that the acid addition salt of formula IIexhibits an X-ray diffraction diagram essentially as in FIG. 2/3 theterm “essentially” means that at least the major lines of the diagramdepicted in FIG. 2/3, i.e. those having a relative line intensity ofmore than 10%, especially more than 20%, as compared to the most intenseline in the diagram, have to be present.

The invention expressly relates also to those forms of themethanesulfonic acid addition salt of a compound of formula I in whichcrystals of the crystal form according to the invention, especially theβ-crystal form, are present in essentially pure form along with othercrystal forms and/or the amorphous form of the4-(4-methyl-piperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]benzamidemethanesulfonate. Preferred, however, is the acid addition salt offormula II, which is present in essentially pure form in the β-crystalform.

The new crystal form, especially the β-crystal form, has the followingproperties:

The melting point in the DSC thermogram of the β-crystal form is 217°C., and that of the α-crystal form is 226° C. (start of melting).

The X-ray diffraction diagram of the β-crystal form does not show thepeak of the α-crystal form marked (1) and only to a very minor extentshows that marked (3) (see FIG. 1/3 and 2/3). By contrast FIG. 2/3 showsa new additional peak marked (4). The new peak marked (5) also appearsin FIG. 2/3.

The X-ray diffraction diagrams also show other marked differences.

In the preferred embodiment, the essentially pure methanesulfonic acidaddition salt of a compound of formula I in the β-crystal form shows theX-ray diffraction diagram indicated in FIG. 2/3.

(i) Preferred is a crystal form of the methanesulfonic acid additionsalt of a compound of formula I which does not show the peak marked (1)in FIG. 1/3 on the X-ray diffraction diagram, this crystal formpreferably being present in essentially pure form.

(ii) Preferred is also a crystal form of the methanesulfonic acidaddition salt of a compound of formula I which remains dry at 93%relative humidity and at a temperature of 25° C., this crystal formpreferably being present in essentially pure form.

(iii) The invention relates preferably to the β-crystal form of themethanesulfonic acid addition salt of a compound of formula I which ischaracterised by the presence of crystals displaying the form shown inFIG. 3/3 below; especially the β-crystal form in essentially pure form.

(iv) Stronger preference is for the β-crystal form of themethanesulfonic acid addition salt of a compound of formula I which hasa melting point of less than 225° C., especially between 217 and 225° C.

(v) Stronger preference is also for the β-crystal form of themethanesulfonic acid addition salt of a compound of formula I which hasa melting point of less than 217° C., defined as the start of melting inthe DSC thermogram.

(v) Stronger preference is also for the β-crystal form of themethanesulfonic acid addition salt of a compound of formula I which onX-ray diffraction shows the peak marked (4) in FIG. 2/3.

(vii) Stronger preference is also for the β-crystal form of themethanesulfonic acid addition salt of a compound of formula I which onX-ray diffraction shows the peak marked (5) in FIG. 2/3.

(viii) Still stronger preference is for the β-crystal form of themethanesulfonic acid addition salt of a compound of formula I whichshows an X-ray diffraction diagram of the type shown in FIG. 2/3,especially one in which the relative peak intensities of each peak donot deviate by more than 10% from the relative peak intensities in thediagram shown in FIG. 2/3, especially an X-ray diffraction diagramidentical to that shown in FIG. 2/3.

(ix) Greatest preference is for the β-crystal form of themethanesulfonic acid addition salt of a compound of formula I which hastwo of the properties named in paragraphs (i) to (viii), greaterpreference being for three of the properties in the said paragraphs,especially all the said properties, and most especially those propertiesdefined as being preferred.

Likewise strongly preferred is a crystal form as defined in one of theparagraphs (i) to (ix) in essentially pure form.

Particularly special preference is for the β-crystal form of themethanesulfonic acid addition salt of a compound of formula I obtainableas described in the Examples.

In all cases, a form of the methanesulfonic acid addition salt of acompound of formula I comprising the corresponding above-mentionedcrystal form is also taken to be meant in a wider aspect of theinvention.

The (preferably essentially pure) β-crystal form is obtainable by

a) digesting another crystal form, especially the α-crystal form, or anamorphous starting material of the methanesulfonic acid addition salt ofa compound of formula I, with a suitable polar solvent, especially analcohol, most especially methanol, or also a ketone (especially in amixture with water, for example water/acetone), typically acetone, aN,N-di-lower alkyl-lower alkanecarboxamide, typicallyN,N-dimethylformamide or -acetamide, or a hydrophilic ether, typicallydioxane, preferably in the presence of some water, or mixtures thereof,in suspension at a suitable temperature, preferably a temperaturebetween 20 and 50° C., for example at about 25° C., or

b) dissolving another crystal form, especially the α-crystal form, or anamorphous starting material of the methanesulfonic acid addition salt ofa compound of formula I, with a suitable polar solvent, such asespecially an alcohol, typically methanol or ethanol, a ketone(especially in a mixture with water, for example water/acetone)typically acetone, a N,N-di-lower alkyl-lower alkanecarboxamide,typically N,N-dimethylformamide or -acetamide, or a hydrophilic ether,typically dioxane, or mixtures thereof, preferably in the presence ofsome water, at a suitable temperature, especially after heating thesolvent, or while warming during the dissolution process, in both casespreferably to 25° C. up to the reflux temperature of the reactionmixture, and then initiating crystallisation by adding a small amount ofthe β-crystal form as seed crystal at a suitable temperature, forexample between 0 and 70° C., preferably between 20 and 70° C.

The educt, the α-crystal form of the methanesulfonic acid addition saltof4-(4-methyl-piperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]benzamide,is obtainable for example by precipitating out the salt from a solutionin a solvent other than an alcohol, such as methanol, and without addinga seed crystal of the β-crystal form.

The above conditions on the selective preparation of the individualcrystal forms are not conclusive. In general, for example, it ispossible to vary parameters such as the weight ratio of themethanesulfonic acid addition salt of a compound of formula I to thesolvent. It is also possible to vary the time needed for the preparationof the β-crystal form, especially when the temperatures are adjusted atthe same time.

One of the advantages of the β-crystal form is especially its morecompact crystal form, which results in substantially more beneficialflow properties and thus in better processability of the methanesulfonicacid addition salt of a compound of formula I in the β-crystal formversus the α-crystal form, for example in the manufacture ofpharmaceutical preparations.

It is true that the α-crystal form of the methanesulfonic acid additionsalt of a compound of formula I is metastable at room temperature.However, the β-crystal form of the methanesulfonic acid addition salt ofa compound of formula I is the thermodynamically stable form at roomtemperature. Greater stability is thus to be expected.

Finally, the β-crystal form is less hygroscopic than the α-crystal formof the methanesulfonic acid addition salt of a compound of formula I, ascan be shown by the following table:

On measurement of the crystal forms up to the point where equilibrium isreached (no further adsorption) in a glass climatic chamber at 25° C.and at the humidities shown below, the following water content valuesare found (the % values for the final water content refer to dryweight): Final water content on adsorption Relative humidity α-crystalform β-crystal form (%) (%) (molar) (%) (molar) 12 0.14 0.05 0.08 0.0233 0.18 0.06 0.10 0.03 46 0.14 0.05 — — 54 0.13 0.04 0.14 0.05 66 0.070.02 0.09 0.03 75 0.49 0.16 — — 85 0.18 0.06 0.16 0.05 93 40 13.1 0.150.05 97 63 20.8 23 7.5 100 — — 37 12

It is shown that, at 25° C., the α-crystal form is hygroscopic andrapidly takes up water so that, at 93% relative humidity, the sample isto some extent present in amorphous form, whereas the β-crystal formremains dry under these conditions. Both crystal forms liquify at 97%relative humidity, but this happens very much more quickly with theα-crystal form than with the β-crystal form.

The lower hygroscopicity is a further advantage for processing andstoring the acid addition salt in the β-crystal form.

The methanesulfonic acid addition salt of a compound of formula I, whichis preferably used in the β-crystal form (hereinafter, themethanesulfonic acid addition salt is always taken to mean the β-crystalform), as well as4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]benzamidein free form, possesses valuable pharmacological properties and may, forexample, be used as an anti-tumour agent, as an agent to treatatherosclerosis, as an agent to treat restenosis, for the prevention oftransplantation-induced disorders, such as obliterative bronchiolitis,and/or for preventing the invasion of warm-blooded animal cells bycertain bacteria, such as Porphyromonas gingivalis.

The phosphorylation of proteins has long been known as an essential stepin the differentiation and division of cells. Phosphorylation iscatalysed by protein kinases subdivided into serine/threonine andtyrosine kinases. The tyrosine kinases include PDGF (Platelet-derivedGrowth Factor) receptor tyrosine kinase.

PDGF (Platelet-derived Growth Factor) is a very commonly occurringgrowth factor, which plays an important role both in normal growth andalso in pathological cell proliferation, such as is seen incarcinogenesis and in diseases of the smooth-muscle cells of bloodvessels, for example in atherosclerosis and thrombosis.

The inhibition of PDGF-stimulated receptor tyrosine kinase activity invitro is measured in PDGF receptor immune complexes of BALB/c 3T3 cells,as described by E. Andrejauskas-Buchdunger and U. Regenass in CancerResearch 52, 5353-5358 (1992). A compound of formula I described in moredetail hereinbefore, such as especially its β-crystal form, inhibitsPDGF-dependent acellular receptor phosphorylation. The inhibition ofPDGF receptor tyrosine kinase is measured in a microtitre ELISA assay(cf Trinks et al., J. Med. Chem. 37, 1015-27 (1994).4-(4-Methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]benzamideand the corresponding methanesulfonate salt inhibit the tyrosine kinaseactivity of the PDGF receptor at an IC₅₀ (concentration at whichactivity is inhibited by 50% compared with the control) of about 120 nMand about 100 nM, respectively.

The inhibition of PDGF makes a compound of formula I also suitable forthe treatment of tumour diseases, such as gliomas, sarcomas, prostatetumours, and tumours of the colon, breast, and ovary.

The methanesulfonic acid addition salt of a compound of formula I alsoinhibits cellular processes involving the so-called stem-cell factor(SCF, also known as the c-kit ligand or steel factor), such as SCFreceptor (kit) autophosphorylation and the SCF-stimulated activation ofMAPK kinase (mitogen-activated protein kinase).

The methanesulfonic acid addition salt of a compound of formula I, suchas especially the β-crystal form thereof, thus inhibits also theautophosphorylation of SCF receptor (and c-kit, a proto-oncogen). MO7ecells are a human promegakaryocytic leukaemia cell line which depends onSCF for proliferation. They are obtained from Grover Bagby, OregonHealth Sciences University, USA. The cells are cultivated in RPMI 1649medium supplemented with 10 FBS and 2.5 ng/ml GC-CMF. GM-SCF and SCF arecommercially available. Serum-deprived MO7e cells are prepared andincubated for 90 min at 37° C. with the test substance before beingstimulated with recombinant SCF for 10 min at 37° C. Identicalquantities of cell lysates are analysed by Western blot usingantiphosphotyrosine antibodies (Buchdunger et al., Proc. Natl. Acad. Sci(USA) 92, 2558-62 (1995)). The immunodecorated proteins are detected bymeans of the ECL Western blotting system from Amersham (Amersham, UK). Acompound of formula I, especially the crystal form of themethanesulfonate salt of formula II, inhibits the autophosphorylation ofSCF-R in the micromolar range.

On the basis of the described properties, the methanesulfonic acidaddition salt of a compound of formula I, such as especially theβ-crystal form thereof, may be used not only as a tumour-inhibitingsubstance, for example in small cell lung cancer, but also as an agentto treat non-malignant proliferative disorders, such as atherosclerosis,thrombosis, psoriasis, scleroderma, and fibrosis, as well as for theprotection of stem cells, for example to combat the haemotoxic effect ofchemotherapeutic agents, such as 5-fluoruracil, and in asthma. It mayespecially be used for the treatment of diseases which respond to aninhibition of the PDGF receptor kinase.

In addition, the methanesulfonic acid addition salt of a compound offormula I, such as especially its β-crystal form C, prevents thedevelopment of multidrug resistance in cancer therapy with otherchemotherapeutic agents or abolishes a pre-existing resistance to otherchemotherapeutic agents. Also regardless of the effect describedhereinbefore, the methanesulfonic acid addition salt of a compound offormula I, such as especially the β-crystal form thereof, may be used toadvantage in combination with other antitumor agents.

Also abl kinase, especially v-abl kinase, is inhibited by4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]benzamideand its methanesulfonate salt. The inhibition of v-abl tyrosine kinaseis determined by the methods of N. Lydon et al., Oncogene Research 5,161-173 (1990) and J. F. Geissler et al., Cancer Research 52, 4492-8(1992). In those methods [Val⁵]-angiotensin II and [γ⁻³²P]-ATP are usedas substrates.4-(4-Methyl-piperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)-pyrimidin-2-ylamino)phenyl]benzamidehere shows an IC₅₀ of 38 nM.

By analogy, the salt of a compound of formula I also inhibits BCR-ablkinase (see Nature Medicine 2, 561-566 (1996)) and is thus suitable forthe treatment of BCR-abl-positive cancer and tumour diseases, such asleukaemias (especially chronic myeloid leukaemia and acute lymphoblasticleukaemia, where especially apoptotic mechanisms of action are found),and also shows effects on the subgroup of leukaemic stem cells as wellas potential for the purification of these cells in vitro after removalof said cells (for example, bone marrow removal) and reimplantation ofthe cells once they have been cleared of cancer cells (for example,reimplantation of purified bone marrow cells).

In addition, the methanesulfonic acid addition salt of a compound offormula I shows useful effects in the treatment of disorders arising asa result of transplantation, for example, allogenic transplantation,especially tissue rejection, such as especially obliterativebronchiolitis (OB), i.e. a chronic rejection of allogenic lungtransplants. In contrast to patients without OB, those with OB oftenshow an elevated PDGF concentration in bronchoalveolar lavage fluids. If4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]benzamidemethanesulfonate, especially in the β-crystal form, is administered torats with tracheal allogenic transplants, for example in a dose of 50mg/kg i.p., it can be shown after removal of 10 transplants per groupafter 10 and 30 days for morphometric analysis of possible epitheliallesions and occlusion of the airways, and investigation forimmunohistochemical pathways of action that, although themethanesulfonic acid addition salt of a compound of formula I has nosignificant effect on epithelial necrosis or infiltration byinflammatory cells, it does markedly reduce fibroproliferation andocclusion of the lumen compared with controls. Synergistic effects withother immunomodulatory or anti-inflammatory substances are possible, forexample when used in combination with ciclosporin, rapamycin, orascomycin, or immunosuppressant analogues thereof, for exampleciclosporin A (CsA), ciclosporin G, FK-506, rapamycin, or comparablecompounds; corticosteroids; cyclophosphamide; azathioprine;methotrexate; brequinar; leflunomide; mizoribine; mycophenolic acid;mycophenolate mofetil; 15-deoxyspergualin; immunsuppressant antibodies,especially monoclonal antibodies for leucocyte receptors, for exampleMHC, CD2, CD3, CD4, CD7, CD25, CD28, B7, CD45, CD58 or their ligands; orother immunomodulatory compounds, such as CTLA4Ig. If CsA (1 mg/kgs.c.), for example, is combined with the acid addition salt of formula I(50 mg/kg), synergism may be observed.

The methanesulfonic acid addition salt of a compound of formula I isalso effective in diseases associated With vascular smooth-muscle cellmigration and proliferation (where PDGF and PDGF-R often also play arole), such as restenosis and atherosclerosis. These effects and theconsequences thereof for the proliferation or migration of vascularsmooth-muscle cells in vitro and in vivo can be demonstrated byadministration of the methanesulfonic acid addition salt of a compoundof formula I and also by investigating its effect on the thickening ofthe vascular intima following mechanical injury in vivo.

The methanesulfonic acid addition salt of a compound of formula I isused in 0.1N HCl or DMSO at a concentration of 10 mM for in vitrostudies. The stock solution is further diluted with cell culture mediumand used in concentrations of 10 to 0.1 μM for the experiments. For invivo administration, the methanesulfonic acid addition salt of acompound of formula I is dissolved for example in DMSO at aconcentration of 200 mg/ml and then diluted 1:20 with 1% Tween in 0.9%saline solution. After sonication, a clear solution is obtained. Thestock solutions are prepared fresh each day before administration. (Thecompound of formula I may also be dissolved simply in deionised waterfor oral administration or in 0.9% saline solution for parenteraladministration). Administration is carried out 24 hours before theoperation. The methanesulfonic acid addition salt of a compound offormula I is administered to rats in one dose of 50 mg/kg i.p. per dayfor the entire observation period. Control rats are given the same doseof substrate. Oral administration is also possible.

Primary cultures of smooth-muscle aorta cells are isolated from 9 to11-day-old DA (AG-B4, RT1 a) rat aorta using a modification of themethod described by Thyberg et al. (see Differentiation 25, 156-67(1983)). The aorta is opened by means of a longitudinal incision and theendothelium carefully removed. The adventitia and the tunica media areseparated, and the tunica media is digested with 0.1% collagenase andDNAse in phosphate-buffered physiological saline for 30 min at 37° C.The cells are centrifuged, suspended in culture medium, and then allowedto grow on plastic vials. The primary cells are used for the experimentsafter passages 2 to 6. Subcultures are kept in DMEM (Dulbecco's ModifiedEagle's Medium), supplemented with 10% fetal calf serum, 2 mmol/mlglutamine, 100 mmol/ml streptomycin, and 100 IU/ml penicillin. Foridentification purposes, the cells are left to grow on glass slidecovers and stained on SMC-α actin (see below).

The migration of smooth-muscle cells is quantified in vitro using aTranswell cell culture insert (Costar, Cambridge, Mass.) whose upper andlower compartments are separated by a polycarbonate membrane of 8 μmpore size. The cells (100 μl at a concentration of 1 million cells/ml)are exposed in the upper compartment. After 2 hours, 60 ng/ml PDGF-BB orPDGF-M (Upstate Biotechnology Inc., Lake Placid, N.Y.) is added to thelower compartment, supplemented with 0.5% fetal calf serum and 0.1%bovine serum albumin, and the test compound is added in concentrationsof 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003 μM. To measurefibronectin-dependent migration, the Transwell chambers are covered withfibronectin at a concentration of 10 μg/ml for 24 h at 4° C. (humancellular fibronectin, Upstate Biotechnology Inc.). After 24 hours'migration, the filters are removed, fixed in methanol, and stained withMayer's haematoxylin and eosin. The migrated cells on the lower side ofthe filter membrane are determined by counting the specified sectionalfields on the filters with the aid of a light microscope with amagnification of 400×. The inhibition of migration is quantified interms of the percentage of cells versus with the control. To exclude thepossibility of a toxic effect, the viability of the cells is tested byincorporation of 3H-thymidine in DMEM, supplemented with 10% fetal calfserum. An inhibition of migration induced by PDGF-M and especially byPDGF-BB is observed.

Experimental animals: the aorta and carotid artery of male Wistar rats(purchased from the Laboratory Animal Center of the University ofHelsinki, Finland) are denuded. The rats are anaesthetised with 240mg/kg chloral hydrate i.p. Buprenorphine (Temgesic, Reckitt & Coleman,Hull, UK) is administered for perioperative and postoperativealleviation of pain. All animals are given human care in keeping withthe “Principles of Laboratory Animal Care” and the “Guide for the Careand Use of Laboratory Animals” of the NIH (NIH Publication 86-23,revised 1985). Rats weighing 200-300 g were used for the denudationprocedure. The left common carotid artery is denuded of endotheliumthrough the intraluminal passage of a 2F embolectomy catheter (BaxterHealthcare Corporation, Santa Ana, Calif., 27). To remove theendothelium, the catheter is passed through the lumen three times,inflated with 0.2 ml air. The external carotid is ligated after removalof the catheter and the wound closed. The histological changes areevaluated by reference to sections of mid-carotid 4 days afterdenudation. The thoracic aorta is denuded of endothelium using a 2FFogarty arterial embolectomy catheter. The catheter is inserted into thethoracic aorta via the left iliac artery, inflated with 0.2 ml air, andpassed through the lumen five times to remove the endothelium. The iliacartery is then ligated. Three times (3, 7 and 14 days) are selected forevaluation of the histological changes.

To quantify the proliferating cells, 3 different procedures are used forlabelling the cells with bromodeoxyuridine (BrdU) after denudation ofthe rat carotid. In this model, the media cell proliferation begins 24 hafter denudation; cells in the intima first appear after 72-96 hours. Toquantify the proliferation of smooth-muscle cells before the appearanceof cells in the intima, 0.1 ml BrdU-labelling reagent (ZYMED, SanFrancisco, Calif.) is administered i.v. during the postoperative periodof 0 to 72 h post-denudation (in total 0.1 ml 6 times). To quantify theproliferation during the initial wave of migration, the rats were given3×0.1 ml BrdU-labelling reagent at 8-hour intervals over a period of72-96 hours after the operation. To quantify the proliferation at theend of the initial wave of migration, a third group of rats is given apulsed dose of 0.3 ml BrdU three hours before sacrifice.

Histological samples are fixed in 3% paraformaldehyde solution for 4 hfor embedding in paraffin. Morphological changes are evaluated fromparaffin sections stained with Mayer's haematoxylin-eosin. The cellcounts of different vessel sections are calculated at a magnification of400×. To identify cells in culture and cells appearing in the neo-intimawithin four days of the denudation injury, immunohistochemical stainingof acetone-fixed samples is carried out using an anti-a-actin antibodyobtained from smooth-muscle cells (Bio-Makor, Rehovot, Israel). Primarysmooth-muscle cells are identified on acetone-fixed glass cover slidesusing the same staining method. The sections are incubated with theprimary antibody (dilution 1:2000), washed, and incubated consecutivelywith peroxidase-conjugated rabbit-antimouse-Ig and goat-antirabbit-Ig,followed by treatment with substrate solution with the chromogen3-amino-9-ethylcarbazol and hydrogen peroxide. BrdU stains are preparedfrom paraffin sections using a primary mouse antibody (Bu20a, Dako, A/S,Denmark) and the Vectastain Elite ABC-Kit (Vector Laboratories,Burliname, Calif.). The sections are deparaffinised and treated bymicrowave at 500 W (2×5 min in 0.1M citrate buffer, pH 6), followed bytreatment with 95% formamide in 0.15M trisodium citrate for 45 min at70° C. Antibody dilutions are prepared according to the manufacturer'sspecifications. The sections are counterstained with Mayer'shaematoxylin and eosin, and positive cells are counted separately forthe initima, media, and adventitia.

In the carotid of treated animals, a significant decrease is found inthe cell count for smooth-muscle cells. The adventitia and the mediashowed a significant reduction in the cell count. As a result of themethanesulfonic acid addition salt of a compound of formula I, a slightdecrease in the absolute number of BrdU-labelled cells is seen in theintima, media, and adventitia during the first two labelling periods(0-72 and 72-96 h), and after 93-96 h a decrease in the number oflabelled cells is seen in all compartments. Decreases in the number ofsmooth-muscle cells are likewise found in the aorta-denuded animals.

According to these findings, the methanesulfonic acid addition salt of acompound of formula I can thus inhibit the proliferation, and especiallythe migration, of vascular smooth-muscle cells.

The methanesulfonic acid addition salt of a compound of formula I,especially the β-crystal form, is also capable of inhibitingangiogenesis. This may be demonstrated as follows: a chamber containingagar (0.8%) and heparin (2 U/ml) with or without growth factor (VEGF 3μg/ml, PDGF 1 μg/ml or bFGF 0.3 βg/ml) is implanted subcutaneously intonormal mice (C57 BL/6). The methanesulfonic acid addition salt of acompound of formula I is administered orally in a dose showing goodanti-tumour activity in a nude mouse xenotransplant model. Dosing isstarted one day before implantation of the chambers. The chambers areremoved after 5 days. The angiogenic efficacy is quantified by measuringboth the vascularised tissue which has grown around the implant and theblood content of this tissue (external blood). The blood is determinedby measuring the haemoglobin. Although the vessels do not grow into theagar, the agar becomes intensely red if an antiangiogenic effect ispresent. If a compound inhibits the increase in blood that is induced bythe growth factor, this is seen as an indication that the compound inquestion is blocking the angiogenic effect of the growth factorconcerned. Inhibition of the weight but not the volume of blood suggestsan effect on the proliferation of fibroblasts. A suppression of thecontrol response suggests an inhibition of wound healing. At an oraldose of 50 mg/kg once daily, the compound of formula I inhibits theangiogenic effect of all three growth factors (VEGF, PDFG, bFGF).

It goes without saying that all the indicated inhibitory andpharmacological effects are also found with the free base,4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]benzamide,or other salts thereof. The present invention relates especially to theβ-crystal form of the methanesulfonic acid addition salt of a compoundof formula I in the treatment of one of the said diseases or in thepreparation of a pharmacological agent for the treatment thereof.

The antiproliferative, especially anti-tumour, activity of themethanesulfonic acid addition salt of a compound of formula I in vivois, for example, described for the treatment of abl-dependent tumours inNature Med. 2, 561-6 (1996).

The invention relates also to a process for the treatment ofwarm-blooded animals suffering from said diseases, especially a tumourdisease, wherein a quantity of the β-crystal form of the methanesulfonicacid addition salt of a compound of formula I which is effective againstthe disease concerned, especially a quantity with antiproliferative andespecially tumour-inhibiting efficacy, is administered to warm-bloodedanimals in need of such treatment. The invention relates moreover to theuse of the β-crystal form of the methanesulfonic acid addition salt of acompound of formula I for the inhibition of the above-mentioned tyrosinekinases, especially PDGF receptor kinase, v-abl kinase, and/or c-kitreceptor kinase, or for the preparation of pharmaceutical compositionsfor use in treating the human or animal body, especially for thetreatment of tumours, such as gliomas, ovarian tumours, prostatetumours, colon tumours, and tumours of the lung, such as especiallysmall cell lung carcinoma, and tumours of the breast or othergynaecological tumours. Depending on species, age, individual condition,mode of administration, and the clinical picture in question, effectivedoses, for example daily doses of about 1-2500 mg, preferably 1-1000 mg,especially 5-500 mg, are administered to warm-blooded animals of about70 kg bodyweight.

The invention relates also to pharmaceutical preparations which containan effective amount, especially an effective amount for prevention ortreatment of one of the said diseases, of the methanesulfonic acidaddition salt of a compound of formula I in the β-crystal form, togetherwith pharmaceutically acceptable carriers which are suitable fortopical, enteral, for example oral or rectal, or parenteraladministration and may be inorganic or organic and solid or liquid.Especially tablets or gelatin capsules containing the active substancetogether with diluents, for example lactose, dextrose, sucrose,mannitol, sorbitol, cellulose, and/or glycerin, and/or lubricants, forexample silica, talc, stearic acid, or salts thereof, typicallymagnesium or calcium stearate, and/or polyethylene glycol, are used fororal administration, Tablets may likewise contain binders, for examplemagnesium aluminium silicate, starches, typically corn, wheat or ricestarch, gelatin, methylcellulose, sodium carboxymethylcellulose and/orpolyvinylpyrrolidone, and, if so desired, disintegrants, for examplestarches, agar, alginic acid, or a salt thereof, typically sodiumalginate, and/or effervescent mixtures, or adsorbents, colouring agents,flavours, and sweetening agents. The pharmacologically active compoundsof the present invention may further be used in the form of preparationsfor parenteral administration or infusion solutions. Such solutions arepreferably isotonic aqueous solutions or suspensions, these possiblybeing prepared before use, for example in the case of lyophilisedpreparations containing the active substance either alone or togetherwith a carrier, for example mannitol. The pharmaceutical substances maybe sterilised and/or may contain excipients, for example preservatives,stabilisers, wetting agents and/or emulsifiers, solubilisers, salts forthe regulation of osmotic pressure, and/or buffers. The presentpharmaceutical preparations which, if so desired, may contain furtherpharmacologically active substances, such as antibiotics, are preparedin a manner known per se, for example by means of conventional mixing,granulating, coating, dissolving or lyophilising processes, and containfrom about 1% to 100%, especially from about 1% to about 20%, of theactive substance or substances.

The following Examples illustrate the invention without limiting thescope thereof. R_(f)-values are determined on TLC plates coated withsilica gel (Merck, Darmstadt, Germany). The ratio of the solvents to oneanother in the solvent systems used is indicated by volume (v/v), andtemperatures are given in degrees celsius (° C.).

Eluents (Gradients):

HPLC Gradient:

0% b) in a) for 20 minutes, then 0%→30% b) in a) for 10 minutes, then30% b) in a) for 5 minutes.

Eluent a): Ion pairing reagent and methanol (420 ml+580 ml)

Eluent b): Ion pairing reagent and methanol (40 ml+960 ml)

Ion pairing reagent: 7.5 g 1-octanesulfonic acid dissolved in about 800ml water, pH value adjusted to 2.5 with phosphoric acid, and dilutedwith water to 1000 ml.

Column: 150×3.9 mm, packed with Symmetry C18 5μ (Waters),pre-equilibrated with eluent a).

Flow rate 1.2 ml/min, UV detection at 267 nm.

EXAMPLES Example 1 Preparation of β-crystal form of4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]benzamidemethanesulfonate—Variant 1

An 11% (w/w) suspension of4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)-pyrimidin-2-ylamino)phenyl]benzamidemethanesulfonate in the α-crystal form is digested in methanol for twodays at about 25° C. The crystals are isolated by filtration on a glassfilter with a G4 frit and dried overnight at room temperature on filterpaper. Smp (by DSC): 217° C. (start of melting).

The starting material,4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]benzamidemethanesulfonate is prepared as follows: 98.6 g (0.2 mol) free4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]benzamide(for preparation see, for example, Eβ-A-0 564 409) is added to 1.4 lethanol. To this beige suspension, 19.2 g (0.2 mol) methanesulfonic acidis added dropwise over a period of 20 minutes. The solution is heatedunder reflux for 20 minutes and then filtered clear at 65° C. Thefiltrate is evaporated down to 50% and the residue filtered off at 25°C. (filter material A). The mother liquor is evaporated to dryness. Thisresidue and filter material A are suspended in 2.2 l ethanol anddissolved under reflux with the addition of 30 ml water. Coolingovernight to 25° C., filtration, and drying at 65° C. until constancy ofweight is achieved result in4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]benzamideas light beige, crystalline mesylate (α-crystal form).

Example 2 Preparation of β-crystal form of4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl]benzamidemethanesulfonate—Variant 2

50.0 g (101 mmol)4-[(4-methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]benzamideis suspended in methanol (480 ml). 9.71 g (101 mmol) methanesulfonicacid and methanol (20 ml) is added, heated to 50° C., activated carbon(5.0 g) added, and the mixture boiled under reflux for 30 minutes,filtered, and concentrated by evaporation. The residue is dissolved inmethanol (150 ml) and inoculated with4-[(4-methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]-benzamidemethanesulfonate (β-modification, a few mg), leading to crystallisationof the product. Drying at 50 mbar and 60° C. leads to4-[(4-methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]benzamidemethanesulfonate, β-modification; R_(f)=0.58 (methylene chloride:ethylacetate:methanol:concentrated aqueous ammonium hydroxidesolution=60:10:30:2); HPLC: t_(ret)=10.2 min.

Example 3 Preparation of β-crystal form of4-(4-methylpiperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl)pyrimidin-3-ylamino)phenyl]benzamidemethanesulfonate—Variant 3

670 g (1136 mmol)4-[(4-methyl-1-piperazin-1-yl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl-2-pyrimidinyl]amino]phenyl]benzamide,α-modification, is heated in methanol (1680 ml). The solution isinoculated at 60° C. with4-[(4-methyl-1-piperazin-1-yl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl-2-pyrimidinyl]amino]phenyl]benzamidemethanesulfonate (β-modification, 55 mg), whereupon the product startsto crystallise. Drying at 50 mbar and 10° C. leads to4-[(4-methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]-benzamidemethanesulfonate, β-modification; R_(f)=0.58 (methylene chloride:ethylacetate:methanol:concentrated aqueous ammonium hydroxidesolution=60:10:30:2); HPLC: t_(ret)=10.2 min.

Example 4 Tablets with4-[(4-methyl-1-piperazin-1-ylmethyl)-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamidemethanesulfonate, β-crystal form

Tablets containing 100 mg of the active substance named in the title areusually prepared in the following composition: Composition Activeingredient 100 mg Crystalline lactose 240 mg Avicel  80 mg PVPPXL  20 mgAerosil  2 mg Magnesium stearate  5 mg 447 mg

Preparation: The active substance is mixed with carrier materials andcompressed on a tableting machine (Korsch EKO, punch diameter 10 mm).

Avicel is microcrystalline cellulose (FMC, Philadelphia, USA).

PVPPXL is polyvinylpolypyrrolidone, cross-linked (BASF, Germany).

Aerosil is silicon dioxide (Degussa, Germany).

Example 6 Capsules with4-[(4-methyl-1-piperazin-1-ylmethyl)-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]benzamidemethanesulfonate, β-crystal form

Capsules containing 100 mg of the compound named in the title as activesubstance are usually prepared in the following composition: CompositionActive ingredient   100 mg Avicel   200 mg PVPPXL   15 mg Aerosil    2mg Magnesium stearate  1.5 mg 318.5 mg

The capsules are prepared by mixing the components and filling themixture into hard gelatin capsules, size 1.

1. A form of the monomethanesulfonic acid addition salt of a compound offormula I,

comprising at least 90% by weight crystals of the β-modification, saidcrystals of the β-modification being non-hygroscopic and remainingessentially dry in a glass climatic chamber at 25° C. and relativehumidities up to and including 93%.
 2. A crystalline form according toclaim 1 of the methanesulfonic acid addition salt of a compound offormula I, which comprises at least 95% by weight crystals of theβ-modification and remains dry at 93% relative humidity and 25° C.
 3. Acrystalline form according to claim 1 of the methanesulfonic acidaddition salt of a compound of formula I, which comprises at least 99%by weight crystals of the β-modification and remains dry at 93% relativehumidity and 25° C.
 4. The β-crystal form according to claim 1 of themethanesulfonic acid addition salt of a compound of formula I, whichcomprises at least 99% by weight crystals of the β-modification and hasa melting point below 225° C.
 5. The β-crystal form according to claim 1of the methanesulfonic acid addition salt of a compound of formula I,which comprises at least 99% by weight crystals of the β-modificationand has a melting point of less than 217° C., defined as the start ofmelting in the differential scanning calorimetry thermogram.
 6. Theβ-crystal form according to claim 1 of the methanesulfonic acid additionsalt of a compound of formula I, which shows on X-ray diffraction a peakat an angle of refraction 2theta of 20°, said peak having a relativeline intensity of 65 as compared to the most intense line in thediagram.
 7. The β-crystal form according to claim 3 of themethanesulfonic acid addition salt of a compound of formula I, whichshows in an X-ray diffraction diagram lines having a relative lineintensity, as compared to the most intense line in the diagram, of 20 ormore at the following angles of refraction 2theta (relative lineintensities given in parentheses): 9.7° (40), 13.9° (26), 14.7° (23),17.5° (57), 18.2° (90), 20.0° (65), 20.6° (76), 21.1° (100), 22.1° (89),22.7° (38), 23.8° (44), 29.8° (23) and 30.8° (20).
 8. The β-crystal formaccording to claim 5 of the methanesulfonic acid addition salt of acompound of formula I, which has a melting point of 217° C., defined asthe start of melting in the differential scanning calorimetry diagram,and which shows essentially the x-ray diffraction diagram shown in FIG.2.
 9. The β-crystal form according to claim 1 of the methanesulfonicacid addition salt of a compound of formula I for use in a process fordiagnostic or therapeutic treatment of the human or animal body.
 10. Apharmaceutical composition, comprising the β-crystal form according toclaim 1 of the methanesulfonic acid addition salt of a compound offormula I and a pharmaceutically acceptable carrier.
 11. Use of theβ-crystal form according to claim 1 of the methanesulfonic acid additionsalt of a compound of formula I for the preparation of a pharmacologicalagent for the treatment of a tumour disease.
 12. Processes for thepreparation of the β-crystal form according to claim 1 of themethanesulfonic acid addition salt of a compound of formula Icharacterised by a) digesting another crystal form or an amorphousstarting material of the methanesulfonic acid addition salt of acompound of formula I with a suitable polar solvent in suspension at atemperature between 20 and 50° C., or b) dissolving another crystal formor an amorphous starting material of the methanesulfonic acid additionsalt of a compound of formula I, in a polar solvent at a suitabletemperature of 25° C. up to the reflux temperature of the reactionmixture, and then initiating crystallisation by adding a small amount ofthe β-crystal form as seed crystal at a temperature between 20 and 70°C.